Fluorescence in situ hybridization (FISH) and digital-imaging analysis significantly enhance the identification of complex or cryptic chromosomal changes in cancer cells. With multiprobe FISH, nonrandom deletions of chromosomes 1, 3, 9, and 11 have been identified in human papillomavirus (HPV)-negative cervical carcinomas--most likely, delineating regions of tumor-suppressor genes. Cumulative chromosomal changes associated with the process of neoplastic transformation of human exocervical cells immortalized by HPV-16 DNA and converted to tumorigenicity by Ha-ras cotransfection was demonstrated by FISH and multicolor detection. The progression towards tumorigenicity was associated with chromosome 1, 3, 9, and 17 anomalies which commonly occur in cervical carcinomas and demonstrate the relevance of in vitro models for studying cervical neoplasia. FISH also substantially increases the resolution of mapping cancer-related genes in mammalian chromosomes. Among several new genes mapped, two histone genes, H2A.Z and H2A.X, were localized to chromosomes 4q24 and 11q23.2-23.3, respectively. The latter site is a highly recombinogenic region in the human genome. A putative multidrug resistance (MDR) enhancerlike element was located 10 kb upstream to the proximal MDR1-P1 promoter on chromosome 7. FISH analysis revealed that this enhancerlike element is on chromosome 20q13.1 and is distinct from the MDR locus on chromosome 7. Using FISH with probes directly biotinylated by polymerase chain reaction, single-copy genes of 200-400 bp were localized on metaphase chromosomes. Reciprocal chromosomal translocations, amplification, direct visualization of multiple members of a gene family, or order of genes within a genomic site were demonstrated using this approach. This new development has unlimited potential for the simultaneous discovery and mapping of new genes, for identification of markers associated with neoplasia and other disorders, as well as for position-cloning strategy.